Renewable Energy 77 (2015) 456e462

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Lipid production from Arundo donax grown under different

agronomical conditions
Domenico Pirozzi a, *, Nunzio Fiorentino b, Adriana Impagliazzo b, Filomena Sannino b,
Abu Yousuf c, Gaetano Zuccaro a, Massimo Fagnano b
a

degli Studi di Napoli Federico II, P.le Tecchio, 80,
Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale (DICMAPI), Universita
80125 Napoli, Italy
b

degli Studi di Napoli Federico II, Via Universita

100, 80055 Portici, NA, Italy
Dipartimento di Agraria, Universita
c
Faculty of Engineering Technology (Program of Energy and Environment), University Malaysia Pahang, Gambang 26300, Malaysia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 30 June 2014
Accepted 19 December 2014
Available online

Hydrolysates of Giant reed (Arundo donax) biomass from three different agronomical conditions were
used to grow the oleaginous yeast L. Starkey. The agronomical conditions affected the cellulose fraction of
biomass, the amount of inhibitors generated during the acid hydrolysis, and the triglyceride yield after
the yeast fermentation. Yet, the composition of triglycerides was not affected.
Different approaches were developed to reduce the effect of inhibitors. The preliminary dilution of
hydrolysates was studied, obtaining the highest values of biomass and lipid yields with a 50% dilution.
Alternatively, the hydrolysates were pre-treated by adsorption and overliming. The latter pre-treatment
gave the best results. A third approach was offered by the use of pre-adapted yeasts, that were able to
grow in the presence of raw hydrolysates.
The composition of the microbial triglycerides was compatible with the production of a biodiesel
suitable for use as automotive fuel.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Arundo donax
Lignocellulose
Yeasts
Lipids
Biodiesel

1. Introduction
Biodiesel is a renewable, biodegradable, non-toxic fuel,
potentially alternative to the petroleum-based diesel. Though a
growing interest has been devoted to biodiesel in the last years,
there are some factors still limiting its market penetration. As a
matter of facts, the feedstock materials used for the so-called 1st
generation biodiesel (i.e. vegetable oils and animal fats) cannot
satisfy the demand for biodiesel at the current rate of consumption. In addition, the relatively high cost of these materials,
accounting for about 75% of the biodiesel price, makes biodiesel
more expensive than the mineral diesel [1]. The highest yields of
seeds (3e5 t ha
1) and oils (1.5e2.5 t ha
1) are achieved growing
the more productive crops (i.e. sunower) in plainy, deep and
fertile soils, mostly in Turkey [2], France [3] and Italy [4], under
conditions of high input (high fertilization rates and full irrigation). This implies relevant socio-economic and ethical

* Corresponding author. Tel.: 39 081 7682284; fax: 39 081 2391800.

E-mail address: dpirozzi@unina.it (D. Pirozzi).
http://dx.doi.org/10.1016/j.renene.2014.12.046
0960-1481/ 2014 Elsevier Ltd. All rights reserved.

questions: gross revenue per hectare of high input sunower

(2e3000 dollars ha
1) is not competitive with the alternative
crops for high fertility environments (15e20,000 dollars ha
1 for
vegetables and fruits) and therefore a biofuel crop expansion in
these lands is not reasonably predictable. In more marginal lands,
the sunower productivity, such as that of other oil crops, is too
low to make protable their cultivation: seed and oil yields fall to
1e2 t ha
1 and 0.5e1.0 t ha
1 respectively, and gross revenue
unlikely overcome 1000 dollars ha
1.Furthermore, considering
the actual increase of world population and the consequent food
demand, is it sustainable and ethically acceptable to threaten
food security by using croplands for biofuel crops in place of food
crops? The use of edible oils to produce biodiesel is threatening
food supplies and biodiversity, causing social and environmental
problems in different developing countries.
In order to overcome these problems, a main requirement is the
availability of cheaper sources of triglycerides, requiring only a
limited use of fertile soil [1]. Lignocellulosic biomasses offer a viable
option to obtain renewable energy, reducing the requirement of
fertile soils. An efcient technology for processing lignocellulosic

D. Pirozzi et al. / Renewable Energy 77 (2015) 456e462

biomasses could open new perspectives as regards the biofuel

production, for different reasons:
- A large range of waste biomasses can be recycled, such as nonfood parts of crops (stems, leaves and husks), forest products,
and also industry wastes (woodchips, skin and pulp from fruit
pressing, etc.);
- Suitable non-food crops (switchgrass, jatropha, miscantus, etc.)
can be cultivated in partially-fertile soils, not used for agriculture, to obtain both vegetable oil (to produce biodiesel according
to the traditional method) and lignocellulosic biomasses for
biofuel production;
- Since cellulose and hemicelluloses are the main component of
plants, the yield of feedstock biomasses per unit area is signicantly increased until 40 t d.m. ha
1 [5].
So far, much attention has been directed towards the exploitation of lignocellulosic biomasses for the production of bioethanol.
In the recent years, a different approach has emerged based on the
use of the lignocellulose hydrolysates to grow oleaginous microorganisms [6e11]. Oleaginous microorganisms are able to produce
more than 20% of their weight in the form of lipids, prevalently
made of triacylglycerols. In particular, oleaginous yeasts are
attracting increasing interest due to their simpler cultural requirements. As a matter of facts, they only require nitrogen limiting
conditions and the presence of a carbon source in excess to accumulate lipids [12e14]. The basic physiology of lipid accumulation in
the oleaginous yeasts has been well studied [15].
An important advantage offered by the application of the
oleaginous microorganisms stems from their ability to produce
aerobically lipids from residual organic matters, with no addition of
expensive nutrients. So far, in order to optimise the cost of the
process, as well as to increase its environmental benet, different
residual materials have been tested as possible nutrients for the
oleaginous yeasts, such as sewage sludge [16], primary efuent
wastewaters [17], sugar cane bagasse [9], tomato waste [18], glycerol [19], olive-mill wastewater [20] or other sources [6,13,14].
Recently, lignocellulosic residues, such as agriculture stover, forest
residues and energy plants, have been increasingly considered as
starting materials for biodiesel production [7e10,21,22].
In this study, we demonstrate that the oleaginous yeast Lipomyces starkeyi can be grown in the presence of lignocellulose
hydrolysates obtained from different samples of Arundo donax
(Giant reed), a widely distributed perennial grass. Currently, the
culms of A. donax represent an interesting source of cellulose for
producing paper [23], second generation ethanol or biopolymers
[24]. Furthermore, the residual lignin content (15e20%) can be
interesting for compost production or other high value materials
such as lignin-based resin coatings and composites [23]. The
A. donax is considered a promising crop for industrial fuel production, due to the high biomass productivity, the ability to be
intensively cultivated and the adaptability to different climatic
and soil conditions. A. donax is known to produce high lignocellulosic biomass yields in marginal lands such as polluted soils [25]
and salinized soils [26]. Perennial crops, such as A. donax, may
guarantee an efcient protection of hilly soils subjected to accelerated erosion [27,28]. This is of particular importance, as the
climate changes are increasing the rain erosivity in the soil tillage
period of annual crops, causing catastrophic soil losses until to
250 t ha
1 [29].
The microbial lipids from L. starkeyi can be used as an alternative
feedstock for the synthesis of biodiesel. Specic attention has been
devoted to the cellular growth inhibitors generated during the acid
pre-treatment production, still hindering a full industrial application of the process [10,21,30]. L. starkeyi are able to degrade

457

carbohydrates in soil and ensilage using extracellular carbohydrolases, and contribute to the biodegradation of herbicides [31].
They have already been used to produce lipids [16,20,22], as they
may offer signicant amounts of microbial oils with little reutilisation of the stored lipids [32].
The proposed approach may allow a more efcient exploitation
of the land area. As a matter of fact, from cultivation of oleaginous
plants commonly used for 1st generation biodiesel (e.g. sunower)
a lipid yield of not more than 1e2 t ha
1 is obtained [2e4]. On the
contrary, the yield of lignocellulosic biomasses obtained from A.
donax is usually 20e40 t ha
1 [5]. Assuming a biomass-to-sugars
yield of 55% [33] and sugar-to-lipid yield of 14% [9], a total lipid
yield of 1.5e3.1 t ha
1 can be estimated. In addition, the cultivation
of A. donax can be adapted to marginal lands [25,26], offering good
yields also with low input cropping systems [5].
2. Materials and methods
2.1. Microorganisms and culture medium
The oleaginous yeasts L. starkeyi were obtained from the
collection of the Dipartimento di Biologia Vegetale of the Perugia
University (Italy). The microorganisms were kept on potato
dextrose agar (Sigma) at T 5  C and cultivated in a synthetic Nlimiting medium, containing (g/L): KH2PO4, 1.0; MgSO4 7H2O, 0.5;
(NH4)2SO4, 2.0; yeast extract, 0.5; glucose, 70.0. The growth was
carried out under aerobic conditions at 30  C on a rotary shaker at
160 rpm (Minitron, Infors HT, Switzerland).
2.2. Lignocellulosic biomasses
A. donax was grown in open eld condition in plainy and hilly
areas of Southern Italy. The led site were:
(a) Torre Lama experimental station, University of Napoli,
(40 370 N, 14 580 E, 30 ma.s.l.), here identied as TL.
(b) Centro Rotary experimental station, University of Napoli,
(40 920 N, 15130 E, 717 ma.s.l.) here identied as CR.
Soil texture was similar (Silty-Clay and poor in organic matter)
in the two sites, with higher carbonate content in CR (Table 1). The
meteorological conditions of the two sites were signicantly
different, with more severe water decit in plainy site (Fig. 1). In TL,
reference evapotranspiration was 2.9 mm/day on the average, total
rainfalls were 810 mm/year, and water decit was measured from
March to September (
617 mm). In CR, reference evapotranspiration was 2.3 mm/day on the average and total rainfalls were
1672 mm/year, and water decit was measured only from May to
August (
137 mm).

Table 1
Soil physico-chemical features of the two sites.

Soil layer (cm)

Sand (%)
Silt (%)
Clay (%)
Texture class
pH
Total carbonate (%)
Organic matter (%)
Nitrogen (g/kg)
C/N ratio
K2O (mg/kg)
P2O5 (mg/kg)

Torre Lama (TL)

Centro rotary (CR)

0e30
46.5
22.3
31.7
SiCL
7.4
0.0
1.24
0.92
7.87
430
32

0e30
37.0
24.3
38.2
SiCL
8.1
16.0
1.09
0.93
6.77
350
19

30e60
41.5
28.5
30.0
SiCL
7.5
0.0
1.11
0.62
10.4
432
25

30e60
36.8
25.0
37.7
SiCL
8.3
17.4
0.95
0.81
6.87
362
13

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D. Pirozzi et al. / Renewable Energy 77 (2015) 456e462

2.5. Lipid extraction and measurement

Methanol (5.0 mL) and chloroform (2.5 mL) were added to
200 mg of dry biomass and vortexed 5 s. Subsequently, the cells
were disrupted for 12 min in an Ultrasonic Homogenizer (Omni
Ruptor 250, USA) at 50% power and 90% pulser. The cells were then
ltered off with Whatman no.1 lter paper. The solvent-lipid
mixture was then placed in 50 mL centrifuge tubes and centrifuged for 10 min at 2000 rpm (Rotanta 460R, Hettich, USA) at 20  C.
The lower layer was then transferred to a pear-shape ask with
Pasteur pipette. Again, 10 mL of 10% (v/v) methanol in chloroform
were added to the residue, a new centrifugation was carried out,
and the lower phase was added to that from the rst extraction. The
solvent in the pear-shape ask was evaporated to dryness (BCHI
Rotavapor R-200, Switzerland) and the extracted weight was nally
recorded after drying at 105  C for 1 h.
2.6. Biomass analysis
The biomass concentration could not be measured by optical
density determination, due to the darkness of the medium. In
order to evaluate the dry biomass weight, the biomass was
centrifuged at 6000 rpm for 10 min and dried at 105  C until
constant weight.
Fig. 1. Monthly values of precipitations (P), evapotranspiration (ET0) and water balance (mm per month) in the cultivation sites.

2.7. Chemical analyses

In TL samplings were made at the harvest (December 2010) of
the 3rd year of crop cycle (planting was made in March 2008) and
two water regimes were compared: rainfed vs. full irrigation (100%
restitution of evapotranspiration).
In CR samplings were made at the harvest (January 2011) of the
8th year of crop cycle (planting was made in March 2004). In order
to obtain realistic results, A. donax plants were grown in the hilly
site only under rainfed conditions (no water resources are available
for irrigation in hilly and mountain croplands of Southern Italy).
In both the sites, transplanting was made from rhizomes with a
plant density of 1  1 m, and nitrogen fertilization was made at the
end of rainy period (April) with a low dose (100 kg N ha
1 from
urea).
2.3. Hydrolysis of lignocellulosic biomasses
Oven-dried stems of A. donax (3 g) were suspended in a 5% (v/v)
solution of H2SO4 (30 ml). Preliminary experiments showed this to
be the optimal acid concentration to maximize the fermentable
sugars yield. When adopting higher concentrations of H2SO4, a
lower yield was obtained, as sugar molecules underwent further
degradation reactions. The biomass suspended in acid was autoclaved at 121  C for 20 min [18], neutralized to pH 6.5 by addition of
saturated KOH solution and ltered with lter paper. When
necessary, the hydrolysate suspension was lyophilized, and subsequently redissolved in fermentation media to obtain the required
concentration of sugars.
2.4. Fermentation in hydrolysates of lignocellulosic biomasses
A xed volume (150 mL) of Arundo donax hydrolysate (ADH) was
inserted, without external organic supplement, in conical ask of
500 mL, and inoculated by 2 mL of microorganism suspension,
obtained dissolving 5 loops of solid culture in 8 mL of physiological
solution. The asks were incubated in an rotary shaker at an
agitation rate of 160 5 rpm and T 30  C.

Cellulose, hemicellulose and lignin were measured according to

the method of Ververis et al. [23].
The fatty acids composition was determined by GC analysis on a
Shimadzu GC 17/3 gas-chromatograph equipped with a ame
ionisation detector, following the method adopted by Li and coworkers [12].
The TOC measurements were carried out with a TOC-VCSH/CSN
(Shimadzu, Japan), upon suitable dilution of a culture medium
sample. The TOC values were obtained subtracting the IC (inorganic
carbon) value from the TC (total carbon) value.
The concentration of reducing sugars was measured following
the method of Nelson-Somogyi [34].
Potential inhibitor compounds were analysed by HPLC
(LC2010c), equipped with a refractive index detector (RID-10A,
Shimadzu, Japan), following the method adopted by Chen and coworkers [21].
2.8. Detoxication of ADH
The biomass growth inhibitors generated in the course of the
hydrolysis were partially removed by overliming, increasing the
pH of the hydrolysate to 10.0 by addition of Ca(OH)2. After 1 h,
the hydrolysate was ltrated under vacuum, acidied to pH 5,5
with 5 M H2SO4 and ltrated again after 1 h for precipitate
removal.
Alternatively, the inhibitors were removed by adsorption. The
hydrolysate was rst neutralized with NaOH to pH 6.5, and then
treated with activated charcoal at a weight ratio of 0.05. The hydrolysates were then incubated overnight at 30  C and 160 rpm,
then ltrated to remove the adsorbent. Finally, pH was adjusted to
6,5 with Ca(OH)2 or 5 M H2SO4.
2.9. Statistical analysis
All experiments have been carried out adopting a minimum of
three replicate tests.

D. Pirozzi et al. / Renewable Energy 77 (2015) 456e462

459

Table 2
A. donax: conditions of treatments and cultivation parameters.
Treatment

Site

TL100w
TL0w
CR

Water

Torre Lama
Torre Lama
Centro Rotary

Temperature

Irrigated
Rainfed
Rainfed

High
High
Low
Average
Signicance

Biomass yield

Basal

Height

Culms

Culms

t ha
1f.w.

t ha
1d.w.

% d.m.

Mm

n. m
2

48.7
50.8
42.7
47.4
n.s.

26.4
24.0
20.9
23.8
n.s.

54.2
47.2
48.2
49.9
n.s.

19.5
19.6
21.3
20.2
n.s.

3.8 a
3.7 a
2.8 b
3.4
0,05

7.5 b
10.8 b
24.5 a
14.3
0,01

85.4
88.9
92.6
89.0
n.s.

Note: different letters indicate differences signicant per P  0.05.

3. Results and discussion

3.1. Biomass yield of A. donax
The cultivation trials of A. donax were carried out in two
different sites (Table 1), following three different treatments, as
shown in Table 2. The results in Table 2 also indicate that the
biomass yield did not change signicantly, conrming the high
adaptability of this species to drought (TL0) and to low temperatures (CR). In the less favourable conditions (CR), the reduction of
plant height was compensated by the increased number of culms
per m2. The A. donax did not show signicant differences between
irrigated (TL100w) and rainfed conditions (CR and TL0w), because
the water decit in the hilly site (CR) during the summer was very
limited, while in the plainy site (TL) the deep root system and the
high water retrieval capacity [35] allowed the water uptake also
from the water table, that in that zone is time-invariant and about
1.0e1.5 m deep. The composition of the biomass samples obtained
from each treatment is reported in Table 3 (columns aec), in terms
of cellulose, lignocelluloses and lignin content.

was reached in the presence of 50% ADH. A poor growth was

observed in the presence of 75% ADH, showing that at this concentration the effect of the inhibitors is still signicant. The concentration of reducing sugars decreased until the complete
exhaustion in the tests carried out with 25% and 50% ADH (Fig. 2b),
whereas the sugar conversion in the presence of 75% ADH was
incomplete. The TOC levels, reported in Fig. 2c, were in all cases
reduced until a nal asymptotic level was reached, that was higher
in the presence of the more concentrated medium. This result indicates that a fraction of the organic components of the hydrolysate
could not be metabolized by the yeasts.
Qualitatively similar proles of biomass, reducing sugars and
TOC were obtained growing hydrolysates from samples TL0w and
CR. (data not shown). The nal values of biomass and lipid concentration of L. Starkeyi obtained using different hydrolysates (after
a 50% dilution) are compared in Table 3 (columns def).
The highest values of both parameters were obtained using the
hydrolysate from the sample TL100w. A possible explanation of this
result stems from the higher cellulose content of this sample (column a in Table 3). However, it is known that both the cellular
growth and the lipid synthesis may be inhibited by some

3.2. Growth of L. Starkeyi in the presence of A. donax hydrolysates

All the samples of A. donax were hydrolysed following a conventional procedure (see Materials and methods). The concentrations of reducing sugars in the A. donax hydrolysates (ADH) were
83,7 g/L, 81,2 g/L and 80,8 g/l respectively for the samples TL100w,
TL0w, CR.
When using raw ADH without any external organic supplement
to culture L. starkeyi, no appreciable cellular growth was observed,
and correspondingly no signicant reduction of TOC and sugars
was detected. This result was clearly due to the higher concentration of inhibitors.
The yeasts were cultured in water mixtures of ADH, containing
ADH fractions of 25%, 50% and 75% respectively. The growth curves
obtained with hydrolysates of the sample TL100w, reported
in Fig. 2a, show that a longer lag phase was recorded as more
concentrated ADH are used. The maximum biomass concentration

Table 3
Effect of different cultivation treatments on the composition of the lignocellulosic
biomass and on the composition of L. starkeyi grown in the presence of the hydrolysate ADH 50%.
Treatment

TL100w
TL0w
CR

Composition of lignocellulosic
biomass

Composition of Lipomyces
starkeyi

(a)
Cellulose,
%

(b)
Hemicell.,
%

(c)
Lignin,
%

(d)
Biomass
conc., g/L

(e) Lipid
fraction,
%

(f) Lipid
conc.,
g/L

46.2
43.8
42.6

23.2
23.5
22.8

16.2
18.9
18.7

9.99
7.81
7.50

19.7
19.5
16.7

197
152
125

ADH 50% water-hydrolysate mixture containing an hydrolysate fraction of 50%.

Fig. 2. Time-proles of different variables in the course of the L. Starkeyi growth, in the
presence of the hydrolysates of the A. donax sample TL100w. (a) Dry biomass, (b)
Reducing sugars, (c) Total Organic Carbon (TOC).

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D. Pirozzi et al. / Renewable Energy 77 (2015) 456e462

Table 4
Concentration (g/L) of potential inhibitors in raw hydrolysates of A. donax.
Compound

Treatment

Acetic acid
Levulinic acid
Formic acid
Furfural
5-HMF
Vanillin
Hydroxybenzaldeyde

TL100w

TL0w

CR

6.22
1.44
1.67
0.1
0.73
0.035
0.056

7.22
1.21
1.80
0.38
1.06
0.041
0.101

7.10
1.23
1.88
0.35
1.13
0.055
0.088

5-HMF 5-hydroxymethylfurfural.

degradation products produced in the course of the hydrolysis

[19,20]. These compounds are generated from the degradation of
sugars (e.g. furfural from xylose and 5-hydroxymethylfurfural),
lignin (e.g. vanillin, syringaldehyde, and 4-hydroxybenzaldehyde)
and other components of the lignocelluloses (e.g. acetic acid from
acetyl groups, formic acid from xylose oxidation, and levulinic acid
from glucose oxidation) [36]. The concentrations of some potential
inhibitors in hydrolysates of different samples of A. donax are reported in Table 4. The data show that all these inhibitors, with the
exception of the levulinic acid, are less concentrated in the hydrolysate of the sample TL100w. Consequently, the higher levels of
biomass and lipid concentration obtained using the sample TL100w
can be explained also taking into account the reduced concentration of inhibitors.

Fig. 3. Growth curves of the L. Starkeyi growth in the presence of hydrolysates from
the treatment TL100w. Preliminary treatments: no treatment (B), overliming (,),
active carbon (), overliming active carbon (C).

observed in the previous tests (see Table 3), and, whatever the pretreatment protocol, are signicantly affected only by the sample of
A. donax used. Even in this case, the hydrolysates obtained from the
sample TLw100 offered better performances both in terms of
biomass concentration and lipid fraction.
3.4. Yeast adaptation to inhibitors

3.3. Pre-treatments to remove inhibitors

In order to remove the degradation products generated by the
hydrolysis treatment, the ADH were preliminary treated following
3 different protocols, namely: (a) overliming treatment with
concentrated Ca(OH)2, (b) neutralization with NaOH, followed by
adsorption on active carbons, (c) overliming followed by neutralization and adsorption.
The biomass concentrationetime proles shown in Fig. 3 were
obtained growing L. starkeyi in the presence of hydrolysates subjected to these protocols. The experimental data demonstrate that
both the adsorption and the overliming allow the biomass growth
in the presence of undiluted hydrolysate. However, the adsorption
with active carbon yielded best results in comparison to the overliming. Coupling both methods did not improve signicantly the
results obtained with active carbons. The proles obtained with
samples TL0w and CR in the same tests were qualitatively similar.
The nal biomass concentrations and triglyceride yields
measured after the growth in the presence of the pre-treated hydrolysates are reported in Table 5(a). These results indicate that the
lipid fractions in the yeasts are substantially similar to those

An alternative method to overcome the effect of inhibitors is

offered by the adaptation of the L. Starkey in the presence of hydrolysates at progressively higher concentration. To this scope,
three consecutive 10-day growth cycles were carried out. After the
rst cycle, carried out in the presence of 50% ADH, the biomass was
collected and partly used to as inoculum for the second cycle, carried out with the 75% ADH. Similarly, the biomass obtained after the
second cycle was used to inoculate a 100% ADH medium. The
experimental results shown in Fig. 4, obtained with sample
TLw100, indicate that the pre-adapted L. Starkey were able to grow
in the presence of 100% ADH (i.e. undiluted raw hydrolysates).
Similar results were obtained with samples TL0w and CR.
The nal values of biomass concentration and triglyceride yield,
obtained with adapted yeasts in the presence of hydrolysates of
different samples of A. donax, are reported in Table 5(b). The lipid
fractions in the yeasts showed a slight increase as subsequent cycles were carried out, though they were mainly affected by the
sample of A. donax used. Again, the best values of biomass concentration and lipid fraction were obtained in the presence of hydrolysates of the sample TLw100.

Table 5
Biomass and lipid concentrations of L. starkeyi.
Biomass conc. g/L

Lipid fraction, %

Lipid conc. g/L

(a) Effect of preliminary treatments of the raw hydrolysate (ADH 100%)AC adsorption with active carbon. OL overliming.
Treatment
AC
OL
AC OL
AC
OL
AC OL
AC
OL
TL100w
4.04
1.51
3.78
19.9
19.7
19.8
80.4
29.7
TL0w
3.56
1.45
3.60
19.6
19.8
19.5
69.8
28.7
CR
3.27
1.56
3.18
16.8
16.6
16.6
54.9
25.9
(b) Effect of subsequent adaptation cyclesof L. starkeyiin the presence of ADH 50% (cycle I), ADH 75% (cycle II), and ADH 100% (cycle III).
Cycle
I
II
III
I
II
III
I
II
TL100w
9.31
6.21
5.34
19.7
19.9
19.9
183
124
TL0w
9.11
5.94
5.20
19.5
19.8
19.9
178
118
CR
8.99
5.97
5.03
16.7
16.8
17.1
150
100
ADH 50%, ADH 75% water-hydrolysate mixtures containing hydrolysate fractions of 50% and 75% respectively.
ADH 100% raw hydrolysate.

AC OL
74.8
70.2
52.8
III
106
103
86.0

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461

4. Conclusions
This study demonstrated that it is possible to produce triglycerides from Arundo donax cultivated in marginal lands, to
reduce the competition with food crops for fertile lands, and to
offer a sustainable way to produce renewable energy (biodiesel) or
building blocks for biopolymers.
The conditions of the agronomical treatments affected the
composition of the lignocellulosic biomass, and eventually the lipid
and biomass yields obtained by yeast grown in the hydrolysates.
Preliminary dilution or pre-treatment of hydrolysates, as well as
yeasts pre-adaptation by progressive increases of the hydrolysate
concentration, were effective in reducing the effect of the microbial
growth inhibitors.
The composition of the microbial triglycerides was suitable to
obtain a biodiesel with reduced tendency to oxidation and good
cold performance.
Fig. 4. Growth curves of the L. Starkeyi during subsequent adaptation cycles: cycle I, in
the presence of ADH 50% (B); cycle II, in the presence of ADH 75% (,); cycle III, in the
presence of ADH 100% (). ADH 50%, ADH 75% water-hydrolysate mixtures containing hydrolysate fractions of 50% and 75% respectively. ADH 100% raw
hydrolysate.

Acknowledgements
This work was supported by the Italian Ministry of the University and of Research (Project EnerBioChem, PON01_1966).

3.5. Triglyceride composition

The fatty acids distribution in the lipids extracted from the
yeasts was measured, using samples of L. Starkey cultured in the
presence of different hydrolysates (Table 6).
In a microbial oil, higher concentrations of unsaturated fatty
acids improve cold properties (as they reduce the cloud point, the
pour point, and the cold lter plugging point). Nevertheless, unsaturated fatty acids reduce the oxidation stability [37]. It has been
suggested that oils with high content of monounsaturated fatty
acids and low in unsaturated and polysaturated fatty acids could
accomplish both these antagonistic requirements [38,39]. However,
a microbial oil containing fractions of saturated and monounsaturated fatty acids that are both close to 50% is considered a
reasonable compromise between oxidative stability and cold ow
properties [40,41]. In this view, the composition of the microbial
oils obtained within this study, containing an oleic acid fraction in
the range 45e50%, and a saturated acids fraction above 40% (as
reported in Table 6), is compatible with the production of a biodiesel offering excellent performances as automotive fuel.
The fatty acid distribution (Table 6) was not signicantly
affected by the agronomical conditions of the A. donax. On the
contrary, the pre-treatment of the hydrolysate, as well as the use of
pre-adapted yeasts, leaded to slight increases in the oleic acid
concentration.

Table 6
Distribution (%) of fatty acids in the triglycerides obtained under different experimental conditions.
Hydrolysed sample

ADH ADH 50%

ADH
ADH
50% (TL100w)
50%
50%
(TL100w) (TL0w) (CR) after active
carbon ads.

Myristic acid C14:0

2
Palmitic acid C16:0
23.7
Palmitoleic acid C16:1 <1
Stearic acid C18:0
16.3
Oleic acid C18:1
46.5
Linoleic acid C18:2
6.5
Linolenic acid C18:3
1.6
Arachidonic acid C20:4 <1

1.8
24.1
<1
16.2
45.8
6.7
1.8
<1

1.9
23.8
<1
16.3
46
6.6
1.8
<1

1.7
22.3
<1
15.8
49.5
6.3
1.5
<1

ADH 100%
(TL100w)
with adapted
L. starkeyi
1.9
22.7
<1
16.0
48.7
6.4
1.5
<1

ADH 50% water-hydrolysate mixtures containing an hydrolysate fractions of 50%.

ADH 100% raw hydrolysate.

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